Studies under this grant have been concerned with elucidation of the mechanisms of sex determination in the vertebrates, and with evaluation of the hypothesis that the molecule bearing serologically-defined H-Y antigen is the determinant of the heterogametic (XY) gonad. H-Y genes have been located on the Y chromosome and on the X; evidence has been provided that development of the XY gonad is secondary to reaction of H-Y and a gonadal receptor; and H-Y minus and H-Y plus classes have been discovered among clinical cases of XY gonadal dysgenesis, of which the latter may be at risk for gonadal tumors. Current research is moving toward evaluation of further theory: that testis-determining structural loci of the mammal are autosomal and regulated by the sex chromosomes; that structural H-Y loci comprise a system of genes of which some are absent in XX males; and that H-Y phenotype is predictive for heterogamety among the vertebrates in general, and for malignancy in cases of XY gonadal dysgenesis in the human. Technical advances are likely to be central to this work. We have devised a sensitive ELISA for detection of H-Y at the cell surface or in solution. The ELISA is based on reaction of biotin-conjugated monoclonal H-Y antibody and a plated antigen source followed by addition of avidinbiotin-enzyme complex and substrate. The result is scored for optical density in an ELISA plate reader. By use of this method, free H-Y has been demonstrated in the supernatant fluids of cultured Sertoli cells and cultured Daudi cells, and cell-surface H-Y has been measured by quantitative absorption in the chicken and mouse. The method will be applied to the study of H-Y in normal populations, and the data obtained will be used as a statistical foundation for the study of H-Y in clinical cases of abnormal sex differentiation and in subjects with aberrant sex chromosome constitutions. Our phylogenetic survey has revealed H-Y antigens in 32 new species and has provided evidence that male and female heterogamety and sex-specific expression of H-Y have evolved several times -- independently in the different species. It is proposed to further extend that survey with emphasis on temperature-controlled sex determination and on systems characterizing transition from male to female heterogamety, the latter evidently governed by a single mutation. The expectation is that analysis of sex determining systems in the different vertebrate classes will provide a comprehensive base for further elucidation of the evolution, genetics and function of the putative inducer molecule.